Study : Approaches for Surveying Cosmic Radiation Damage in Large Populations of Arabidopsis thaliana Seeds- an Antarctic Example


Approaches for Surveying Cosmic Radiation Damage in Large Populations of Arabidopsis thaliana Seeds- an Antarctic Example
The Cosmic Ray Exposure Sequencing Science (CRESS) payload system is a proof of concept experiment to assess the genomic impact of space radiation on seeds. CRESS was designed as a secondary payload for the December 2016 high-altitude, high-latitude, and long-duration balloon flight carrying the Boron And Carbon Cosmic Rays in the Upper Stratosphere (BACCUS) experimental hardware. Investigation of the biological effects of Galactic Cosmic Radiation (GCR), particularly those of ions with High-Z and Energy (HZE), is of interest due to the genomic damage this type of radiation inflicts. The biological effects of upper-stratospheric mixed radiation above Antarctica (ANT) were sampled using Arabidopsis thaliana seeds and were compared to those resulting from a controlled simulation of GCR at Brookhaven National Laboratory (BNL) and to laboratory control seed. The payload developed for Antarctica exposure was broadly designed to 1U CubeSat specifications (10cmx10cmx10cm, ≤1.33kg), maintained 1 atm internal pressure, and carried an internal cargo of four seed trays (about 580,000 seeds) and twelve CR-39 Solid-State Nuclear Track Detectors (SSNTDs). The irradiated seeds were recovered, sterilized and grown on Petri plates for phenotypic screening. BNL and ANT M0 seeds showed significantly reduced germination rates and elevated somatic mutation rates when compared to non-irradiated controls, with the BNL mutation rate also being significantly higher than that of ANT. Genomic DNA from mutants of interest was evaluated with whole-genome sequencing using PacBio SMRT technology. Sequence data revealed the presence of an array of genome structural variants in the genomes of M0 and M1 mutant plants. Overall design: Arabidopsis thaliana seeds of the Columbia (Col-0) ecotype were placed into 24 x 24 bin seed trays (modified 1536-well plates) with bins which were then covered by polyolefin sealant film [USA Scientific] and flanked by CR-39 SSNTDs, for two non-concurrent exposures. In the BNL exposure, one seed tray with three CR-39 sheets on each side was placed in a modified T-25 flask for irradiation (H, 250 MeV, 10.62 Rad; He, 250 MeV, 3.165 Rad; O, 350 MeV, 1.08 Rad; Ti, 300 MeV, 0.735 Rad). The Antarctica exposure was 30 days in duration at an altitude of ~35 km. Four seed trays were arranged in a stack with three CR-39 plates at each end, and two sheets between each tray. After irradiation, seeds were returned to the University of Florida and stored at -20 °C until planting. Seeds were planted on Phytagel™-based nutrient media and grown for 14 days, at which time Petri plates were evaluated for germination and mutation rates. Mutants of interest were tracked, and preserved in liquid nitrogen if growth was deemed too impaired for the plant to mature. Whole genomic DNA was extracted from 10 mutants, which was used to prepare SMRT-Bell libraries (4 individual mutants, 1 pool of 6 mutants). The PacBio Sequel was used for Whole-Genome Sequencing of the samples, and the output sequences were processed to detect structural variants using the NGMLR and Sniffles softwares.


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